Short guide to circular soil chromatography

Acknowledgements

This booklet was produced in the framework of the project series
“UROS - Ubiquitous Rural Open Science Hardware” (1), a collaboration
of the Global Hackteria Network and mikroBIOMIK Society (2),

Humus Sapiens, Gathering for Open Science Hardware (3) and Ayllu

Cooperativa (4). Zavod Rizoma (5) has offered the research location
during the “Maribor soil week“ in Mai 2022.

The UROS project has been supported financially as part of the @&)

konS = Platform for Contemporary Investigative Arts (6), a project

chosen on the public call for the selection of the operations

“Network of Investigative Art and Culture Centres”. The investment

is co-financed by the Republic of Slovenia and by the European SBIOMIK
Regional Development Fund of the European Union.

Links UMUS SAPIENS

(1) hackteria.org/wiki/UROS

(2) mikrobiomik.org/humussapiens
(3) openhardware. science

(4) instagram.com/ayllucoope

(5) zavodrizoma.si

(6) kons-platforma.org

8 REPUBLIC OF SLOVENIA

Text & images MINISTRY OF CULTURE

Julian Chollet,
Fernando “Nano” Castro WS
£3 | EUROPEAN UNION
EUROPEAN REGIONAL
° . . DEVELOPMENT FUND
Design & illustration WA
Akvilé PaukStyté

Created 09/2022
Modified 12/2022

Download
the digital version

at archive.org or (cc) (#) (9)
mikrobiomik.org Published under CC BY-SA 4.0 BY

Introduction

Soil is an extremely complex substance: minerals, water,
air, organic material and a multitude of living beings come
together to create a dynamic self-regulating system. For
the analysis of soil properties, many different approaches
can be useful. Physical characteristics like aggregation
and pore structure, can inform us about water retention
capacities and possible problems with compaction;

the chemical composition informs us about the lack

and abundance of certain minerals and the biological
diversity allows us certain assumptions concerning the
overall function of the soil food web and therefore on the
capability of the soil to support plant growth.

Circular chromatography of soil extracts is a method

of analysis that was developed in the mid- to late 20th
century and is still used by “biodynamic” farmers all
over the world. Although it’s scientific validity is being
disputed, the procedure follows a strict protocol and
yields highly reproducible results. Similar to other
qualitative approaches, personal experience is the key to
obtain valuable information and with some practice this
method might allow insights that reach beyond classical
physical and chemical analysis. Due to it’s simplicity

and the aesthetic value of the chromas, circular soil
chromatography is also highly suited for education and
as a tool to reconnect farmers, gardeners and the general
public to the soil on which their life depends.

Further readings

Pfeiffer, E.E. (1960) “Qualitative chromatographic method for
the determination of biological factors” Biodynamics 50, 2-15.

Hassild-Piezunka (2003) Eignung des Chroma-Boden-Tests zur
Bestimmung von Kompostqualitat und Rottegrad. Ph.D. Dissertation,
Carl-von-Ossietzky University Oldenburg

William F Brinton “Assessing Compost & Humus Condition by Circular
Chromatography” (2010) Journal of the woods end research Lab Vol 1:1

Restrepo Rivera, J. R; Pinheiro, S. (2011) “Cromatografia -
Imagenes de vida y destrucci6én del suelo” Juquira Candiru Satyagraha

Maria Olga Kokornaczyk, Fabio Primavera, Roberto Luneia, Stephan
Baumgartner & Lucietta Betti. (2016) “Analysis of soils by means
of Pfeiffer’s circular chromatography test and comparison to

chemical analysis results” Biological Agriculture & Horticulture.

Ford, B., Cook, B., Tunbridge, D., and Tilbrook, P. (2019)

“Using paper chromatography for assessing soil health in southwestern
Australia” Centre of Excellence in Natural Resource Management,
University of Western Australia.

Benjamin M. Ford, Barbara A. Stewart, David J. Tunbridge,
Pip Tilbrook, (2021) “Paper chromatography: An inconsistent tool
for assessing soil health” Geoderma, Volume 383,

7

Some examples

Agricultural soils

Non cultivated soil
(next to grape farm)

Conventional grape farm

Regenetative grape farm
(3 years after transition)

The 3 images on the side show the chromas of samples
that were taken in close proximity to each other in the wine
growing region of Mendoza, Argentina. The first represents
non-cultivated soil; the second a conventional grape farm
that uses chemical fertilizers and pesticides; the third a
“regenerative” organic grape farm 3 years after transition.

The non-cultivated soil sample shows a very light central
zone, clearly separated concentric rings and a slightly fuzzy
outer edge. The sample from the conventional grape farm also
shows a clear separation of the zones, an even more fuzzy
edge, but a much darker coloring in the central zone. The
chroma of the regenerative grape farm is quite different from
the first two, as it shows blurred zone borders and a defined
outer edge with a dark rim. All chromas lack radial features.

The main differences between the 3 chromas are:

1) coloring; 2) zone separation; 3) outer edge.

1) Dark brown colors are usually associated with humus
content. The soil from both grape farms shows a higher
amount of organic compounds than the non-cultivated soil,
but their distribution is different. The regenerative farms
sample seems to lack certain large organic molecules (light
patch in the central zone) but has a higher amount of small
organic molecules (very dark outer zones).

2) We could not find any conclusive explanation for the blurry
zone borders in the regenerative farms sample. It could be
speculated that a complex soil-food-web creates a high
diversity of organic and inorganic compounds, which then
leads to a more diffuse appearance on the chroma, but we
have no evidence to support this idea.

3) A defined outer edge is usually considered a sign of high
soil fertility. It is associated with strong microbial activity and
the presence of small organic molecules.

How it works

Liquids travel through filter paper, drawn by capillary forces.
The individual components of mixed samples ‘migrate’

faster or slower according to their size and physical/
chemical properties. This specific way of separation is called
chromatography.

In circular soil chromatography, the extracts are made with
sodium hydroxide - a substance widely used to extract organic
matter from soil and compost samples. It reacts actively,
breaking down rigid, solid substances, long and complex
molecules, making them smaller and more mobile. Before
applying the soil extracts, the filter papers are soaked with

a very diluted solution of silver nitrate, which is known for its
extreme sensitivity to light. The soil components that are being
separated by the filter paper create specific patterns and
when they react with silver nitrate, some of them also develop
characteristic colors.

Similar substances share similar characteristic patterns and
colors, which means that soil samples from ‘conventional’
industrial agriculture are similar to each other but very
different from rich organic soils or composts. Over the last
decades, some efforts have been made to quantify and
objectify the results of soil chromatography, with interesting
results (see chapter X & further reading) but it’s main strengths
may lie precisely in it’s subjective nature.

high precision is not needed if you

- scale (at least 0.1g precision) 7 have the AgNO, as a solution
- measuring glass (~50-100ml)

- glass jars (min. 100ml)

- petridishes, lids of jars, or something similar

- pipette (~2-10 ml)

- rubbergloves

- scissors

(for 20 chromas)

Materials

- silver nitrate (AgNO,) + 0,29

- sodium hydroxide (NaOH) —>» 10g

- distilled water =F ~1.5 | SOF -Ehen wid: be used

- 22 filter papers (15 cm diameter) for the “wicks” (see step 3)

Plan in advance:

AgNO, and suitable filter papers may have 1-2 weeks delivery time.

The other materials are usually available in any drug store.

Filter papers: we had good experiences with retention rates of 5-8 um -

but others may work fine as well.

The workflow

If you do chromatography for the first time, we recommend to start with only few
samples to get a feeling for the process and concentrate on adapting the workflow to
your local conditions. Try a few very different soils, to get a glimpse on the variety of
shapes/colors and compare the same sample in two different dilutions. Take your time
to observe and get some practice with handling the material. Whenever you are ready
for a more systematic approach, begin by formulating a precise research question:
e.g. is there a difference between the part of the garden that you covered with mulch

and the part that you left exposed?

Recommendations:

- make a time-plan - when to take samples, prepare the filter-paper, etc.
- find a place where you can improvise a darkroom (see step 3)

- take at least 2 samples of each plot that you want to compare

- make a “blank” chroma with pure extraction solution (1% NaOH)

- be very precise in the preparation — treat all samples exactly the same

Some examples
Sand vs. soil

100% sand

75% sand / 25% garden soil

50% sand / 50% garden soil

The evaluation of soil quality is a highly
complex problem and circular chromatography
might help to understand certain aspects, but
since we are no experts on this technique
(yet), we do not want to claim that the
following interpretations are correct.

We generally recommend a combination of
several methods, including visual & haptic
(e.g. as described by Graham Shepherd) and
microbiological approaches (see also our
“Short guide to soil microscopy”).

The 3 images on the side show how circular chromatography
reacts on different ratios of sand and garden soil. For the
experiment we used sand from a construction site and rich,
dark brown soil from a mulched garden bed. The results
were highly reproducible and consistent with our previous
experiences concerning color saturation and zone formation
in soils with different humus content.

Circular chromatography is highly sensitive: with 50% garden
soil (2,5 g in 50 ml NaOH) the chroma was fully saturated.

A further increase of concentration did not produce any
change in color, zoning or radial features (images not shown).
The samples with pure sand were pale, showed a gradient
instead of zones and had a fuzzy edge. According to literature,
this can be attributed to the very low amount of organic
compounds and weak microbial activity. Even small amounts
of garden soil lead to a clear separation of zone borders,

a defined edge without spikes and strong radial features
(,channels“) that penetrated most of the zones. Except for

the color intensity, there are two main differences between

25 % and 50 % garden soil: 1) the thin light rim on the outer
edge of the chroma, which was only visible in the lower
concentrations; 2) the number, shape & size of the channels,
which were fewer but broader and reaching into the central
zone, only in the higher concentrations.

We conclude from these experiments, that the optimal dilution
for chromatography has to be determined individually for
each sample. Otherwise it might happen that important
differences get lost due to saturation or other concentration
dependent effects in pattern formation.

Interpretation ez evaluation

An image is worth more
than a thousand numbers

Chromas are unique, beautiful images that reveal some of the complexity and
integrity of the soils they originate from. We like the method because it is simple
enough to be performed at home or ina workshop and it allows us to capture some
of the properties of a soil sample onto a piece of paper that we can admire, share
and archive (e.g. on the refrigerator). Most importantly, the image is not created by
us, but by the chemical and physical composition of the soil and by the billions of
microorganisms that inhabit it. The patterns and colors directly emerge from the
soil’s living system - we can only assist in their manifestation and development.

Within the framework of biodynamic agriculture, circular chromatography is not
only considered a reliable method for analyzing soil composition, but the chromas
are also interpreted in regards of the ‘energetic’ properties of the soil and their
ability to support plant life. On the following pages, we want to share the insights
from two of our experiments, but we will not dive deeper into the art of ‘reading’ a
soil chroma. If you want to learn more about how to interpret the results, please
consult scientific publications on the topic (see “further reading”) and/or contact
your local biodynamic farming association.

People with great experience have standardized the reading by separating the
chroma into 3-5 concentric rings (zones) and other features, like spikes and
channels. The presence and colors of those zones and their relation to the radial
features has allowed them to distinguish and characterize properties like: the
maturity of a compost (Binton, 2010), the presence of industrial practices (e.g.
use of chemicals & heavy machinery) and the effectiveness of using compost
(Restrepo and Pinheiro. 2011).

It is evident that for a better interpretations of a chroma we must conjugate a certain
amount of experience with the method, a basic knowledge of the chemistry and
physics of the process involved but most importantly, we need to appeal to our
experience and knowledge of the samples, its smell, texture, history: where is it
from? what was grown here? what kind of treatment did it receive over the years?

STEP I -
Preparing the solutions

minimize light exposure when handling AgNO,!!

0.5 % AgNO, solution 7
e.g. 0.5g AgNO, in 100ml of distilled water
you will need 2 ml per chroma, so this would be enough

for about 5@ chromas - you can store the solution in a
lightproof bottle (use aluminum foil to improvise)

1% NaOH solution
e.g. 10g NaOH in 11 of distilled water

you will need 5@ ml per chroma, so this is enough for
2@ chromas - no special storage conditions needed

STEP 2 —
Making soil extracts

- collect a handful of soil (without big stones, roots, plants, etc.)

- if you want to compare locations, take at least 2 samples from each place

- spread the soil on a table/cardboard/etc. to let it dry —» tidak langsung di bawah matahari
- sieve 5 g of dry soil (~1-2 mm holes) —+» some protocols use 10 g,

- mix the soil with 50ml 1% NaOH DUE “For: US) ig ‘Worked ‘better

- gently shake or stir the solution several times during the next 2-3 hours —> é

- let the sample settle/ sediment for 2 h before chromatography at the beginning,
after 15min,
after 1 hour,

50 @ after 2 hours
41% NaOK

te

If the sample is rich in organic
matter it will take longer to settle

Ideally the sedimentation happens in your
‘darkroom’, so you don’t have to move them
again when doing the chromatography

STEP 3 —-

STEP 4 -
I the filt with
rT ee Soaking the filter paper
3

% this step should be done with gloves * AgNO, is a strong stain and can with your soil extracts

and in relative darkness © cause skin irritations

* Keep the filter papers clean 3 this step should be done with gloves

and in relative darkness ©
- improvise a darkroom
close the curtains, hang some
blankets,.. the darker the better F ‘
dit don": pantie sibel Oe. fou - mark the edge of each filter papers (soaked in AgNO.)
can also wait for sunset and use - make a hole in the middle of each filter paper with the sample name (e.g. numbers)
red flashlights or any other low

sueeeatie lieiesuce: to find the middle, you can fold it, mark

the center with a short line and fold - clean the petridishes / jar-lids and use fresh wicks
it again in the other direction - then
you can stack the filters and punch a

- pipette 2 ml of the supernatnat (above the sedimented soil
hole through all of them with scissors aa P ( )

or a knife; gently widen the without mixing the liquid
hole to 3-5 mm size
Pa a) - repeat the same process as for soaking with AgNO,

- make twice as many wicks
as you have filters (you will A3NO, - stop the chromatography when the solution reaches
need them for step 4) ~3 cm from the edge,
cut one filter paper into — or when the image does not change anymore

~2x2 cm squares and roll depending on your sample and paper,

=> we ea c

them into cylinders this can take up to 1h
- pipette 2 ml of AgNO3
solution onto the petridish / - let your chromas dry and then expose them to
: ingaciawiek into ihe hole jar cap indirect sunlight for 2-3 hours

for more controlled conditions, you can use

of the filter paper and artificial light in a darkened environment

place it onto the dish

make sure the wick touches
the solution - it will
immediately start to soak into
the filter paper ~

- repeat this process for all Troubleshooting

filter papers

a

whan ihe sohillon reaches <9 om gain can dealer ahen an - if the solution travels less than ~1/2 the - if all chromas appear pale
from the edge, remove the wick parallel - depending on the distance to the edge, consider using a and without clear patterns,
and let them dry paper it will take 20-30min higher dilution for your next experiments try a higher concentration
~3em place them on some toilet oe aene and/or a longer time for sedimentation of soil (e.g. 10 g)

paper or cardboard and
leave them in the dark